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Abstract:

A method of forming an array of viable cells is carried out by ink-jet
printing a cellular composition containing said cells on a substrate. At
least two different types of viable mammalian cells are printed on the
substrate, the at least two different types of viable mammalian cells
selected to together form a tissue. In some embodiments at least three or
four different viable mammalian cells are printed on the substrate, the
cells selected to together form a tissue. In some embodiments one of the
viable mammalian cell types is a stem cell. In some embodiments the
method further comprises printing at least one support compound on the
substrate, the support compound selected to form a tissue together with
said cells. In some embodiments the method further comprises printing at
least one growth factor on the substrate, the growth factor selected to
cause the cells to form a tissue.

Claims:

1. In a method of forming an array of viable cells by ink-jet printing a
cellular composition containing said cells on a substrate, the
improvement comprising:printing at least two different types of viable
mammalian cells on said substrate, said at least two different types of
viable mammalian cells selected to together form a tissue.

2. The method of claim 1, wherein at least three different viable
mammalian cells types are printed on said substrate, the cells selected
to together form a tissue.

3. The method of claim 1, wherein at least one of said viable mammalian
cell types is a stem cell.

4. The method of claim 1, further comprising printing at least one support
compound on said substrate, said support compound selected to form a
tissue together with said cells.

5. The method of claim 1, wherein said tissue is selected from the group
consisting of nerve, skin, pancreatic islet, and bone tissue.

6. The method of claim 1, wherein said tissue is skin tissue.

7. The method of claim 1, wherein said tissue is bone tissue.

8. The method of claim 1, wherein said tissue is pancreatic islet tissue.

9. The method of claim 1, wherein said tissue is nerve tissue.

10. In a method of forming an array of viable cells by ink-jet printing a
cellular composition containing said cells on a substrate, the
improvement comprising:printing viable stem cells on said substrate.

12. A method of claim 1, further comprising the step of implanting said
array in vivo in a subject in need thereof.

13. The method of claim 12, further comprising maintaining said array in
vivo in said subject for at least one month, during which all cell types
in said array maintain their structural and spatial orientation in vivo.

14. The method of claim 12, further comprising maintaining said array in
vivo in said subject for at least two months, during which all cell types
in said array maintain their structural and spatial orientation in vivo
and retain their cellular characteristics and tissue function.

15. In a method of forming an array of viable cells by ink-jet printing a
cellular composition containing said cells on a substrate, the
improvement comprising:printing viable cancer cells on said substrate.

16. The method of claim 15, wherein said cancer cells are selected from
the group consisting of leukemia, lymphoma, breast, lung, colon,
prostate, ovarian, skin, melanoma, and brain cancer cells.

Description:

[0002]Living tissues maintain an inherent multi-cellular heterogeneous
structure. Rebuilding of such complex structure requires subtle
arrangements of different types of cells and extracellular matrices (ECM)
at their specific anatomical target sites. To achieve tissue
reconstitution, an effective method for a precise delivery of cells and
biomaterials is needed. The inkjet printing technology has been applied
to address this endeavor.

[0007]Although the capability of inkjet printing of viable single cells
has been verified, the possibility of simultaneously printing multiple
cell types to build viable heterogeneous cellular constructs has not been
demonstrated to date. It has been found that distinct cell types can be
mixed with support compounds (collagen gels) and printed into the target
areas to form 3-dimensional tissue structures. Further, basic
physiological functions and properties of each cell type within the
structure can be maintained.

[0008]A first aspect of the invention is, in a method of forming an array
of viable cells by ink-jet printing a cellular composition containing
said cells on a substrate, the improvement comprising printing at least
two different types of viable mammalian cells on said substrate, said at
least two different types of viable mammalian cells selected to together
form a tissue. In some embodiments at least three or four different
viable mammalian cells are printed on said substrate, the cells selected
to together form a tissue. In some embodiments one of said viable
mammalian cell types is a stem cell. In some embodiments the method
further comprises printing at least one support compound on said
substrate, said support compound selected to form a tissue together with
said cells. In some embodiments the method further comprises printing at
least one growth factor on said substrate, the growth factor selected to
cause the cells to form a tissue.

[0009]Example tissues, or tissue substitutes, that may be produced by the
processes of the invention include nerve, skin, pancreatic islet, and
bone tissue.

[0010]Since it is preferred to print three-dimensional arrays when forming
tissues or tissue substitutes as described above, and since such printing
may require substantially greater times than required in prior
techniques, it is sometimes preferred to carry out the printing in a
culture chamber or an environmentally controllable chamber to enhance the
survival of cells after printing.

[0011]Another aspect of the invention is a method of forming an array of
viable cells by ink-jet printing a cellular composition containing said
cells on a substrate, the improvement comprising: printing viable stem
cells (for example, amniotic fluid stem cells) on the substrate.

[0012]Another aspect of the invention is a method of forming an array of
viable cells by ink-jet printing a cellular composition containing said
cells on a substrate, the improvement comprising: printing viable cancer
cells on said substrate. Arrays produced by such methods are useful in
screening compounds for efficacy in treating cancer by contacting the
compound to the cancer cells. Arrays can be printed with normal or
"control" cells adjacent to the cancer cells, so that the test compound
may be concurrently contacted to the control cells, so that the
differential effect of the test compound on cancer cells as compared to
control cells may be evaluated.

[0013]The foregoing and other objects and aspects of the present invention
are explained in greater detail in the drawings herein and the
specification set forth below.

[0018]"Support compound" as used herein may be any naturally occurring or
synthetic support compound, including combinations thereof, suitable for
the particular tissue or array being printed. In general the support
compound is preferably physiologically acceptable or biocompatible.
Suitable examples include but are not limited to alginate, collagen
(including collagen VI), elastin, keratin, fibronectin, proteoglycans,
glycoproteins, polylactide, polyethylene glycol, polycaprolactone,
polycolide, polydioxanone, polyacrylates, polysulfones, peptide
sequences, proteins and derivatives, oligopeptides, gelatin, elastin,
fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides,
polycarbonates, polyanhydrides, polyamino acids carbohydrates,
polysaccharides and modified polysaccharides, and derivatives and
copolymers thereof (see, e.g., U.S. Pat. Nos. 6,991,652 and 6,969,480) as
well as inorganic materials such as glass such as bioactive glass,
ceramic, silica, alumina, calcite, hydroxyapatite, calcium phosphate,
bone, and combinations of all of the foregoing.

[0020]"Cells" as used herein may be of any suitable species, and in some
embodiments are of the same species as the subject into which tissues
produced by the processes herein are implanted. Mammalian cells
(including mouse, rat, dog, cat, monkey and human cells) are in some
embodiments particularly preferred.

[0021]"Stem cell" as used herein refers to a cell that has the ability to
replicate through numerous population doublings (e.g., at least 60-80),
in some cases essentially indefinitely, and to differentiate into
multiple cell types (e.g., is pluripotent or multipotent).

[0022]"Embryonic stem cell" as used herein refers to a cell that is
derived from the inner cell mass of a blastocyst and that is pluripotent.

[0023]"Amniotic fluid stem cell" as used herein refers to a cell, or
progeny of a cell, that (a) is found in, or is collected from, mammalian
amniotic fluid, mammalian chorionic villus, and/or mammalian placental
tissue, or any other suitable tissue or fluid from a mammalian donor, (b)
is pluripotent; (c) has substantial proliferative potential, (d)
optionally, but preferably, does not require feeder cell layers to grow
in vitro, (e) optionally, but preferably, specifically binds c-kit
antibodies (particularly at the time of collection, as the ability of the
cells to bind c-kit antibodies may be lost over time as the cells are
grown in vitro).

[0024]"Pluripotent" as used herein refers to a cell that has complete
differentiation versatility, e.g., the capacity to grow into any of the
animals cell types. A pluripotent cell can be self-renewing, and can
remain dormant or quiescent with a tissue. Unlike a totipotent cell
(e.g., a fertilized, diploid egg cell) a pluripotent cell cannot usually
form a new blastocyst.

[0025]"Multipotent" as used herein refers to a cell that has the capacity
to grow into any of a subset of the corresponding animals cell type.
Unlike a pluripotent cell, a multipotent cell does not have the capacity
to form all of the cell types of the corresponding animal.

[0026]"Cancer cells" as used herein may be of any type, including but not
limited to leukemia, lymphoma, breast, lung, colon, prostate, ovarian,
skin, melanoma, and brain cancer cells.

[0027]Subjects that may be implanted with constructs or arrays of the
present invention include both human subjects and animal subjects
(particularly mammalian subjects such as dogs, cats, horses, pigs, sheep,
cows, etc.) for veterinary purposes.

[0028]The disclosures of all United States patent references cited herein
are to be incorporated herein by reference in their entirety.

A. Tissue Printing.

[0029]Methods and compositions for the ink-jet printing of viable cells
are known and described in, for example, T. Boland et al., US Patent
Application No. 2004/0237822 (Dec. 2, 2004)(Clemson University) and W.
Wilson and T. Boland, The Anatomical Record Part A, 272A: 491-496 (2003).
While the present invention is primarily concerned with ink-jet printing
of cells, the cells may be printed by other means as well, such as the
methods and compositions for forming three-dimensional structures by
deposition of viable cells described in W. Warren et al., U.S. Pat. No.
6,986,739 (Sciperio Inc.).

[0030]Although not required, cells can typically be printed in the form of
a "cell composition" that contains a liquid carrier for the cells. The
cell composition can be in the form of a suspension, solution, or any
suitable form. Examples of suitable liquid carriers include, but are not
limited to, water, ionic buffer solutions (e.g., phosphate buffer
solution, citrate buffer solution, etc.), liquid media (e.g., modified
Eagle's medium ("MEM"), Hanks' Balanced Salts, etc.), and so forth. For
instance, the use of a liquid carrier in the cell composition can ensure
adequate hydration and minimize evaporation of the cells after printing.
However, the probability of obtaining viable cells in any given printed
drop also decreases with decreasing cell concentration. (T. Boland, US
Patent Application Publication No. 20040237822 at para 48)

[0031]Various mechanisms may be employed to facilitate the survival of the
cells during and/or after printing. Specifically, compounds may be
utilized that "support" the printed cells by providing hydration,
nutrients, and/or structural support. These compounds may be applied to
the substrate using conventional techniques, such as manually, in a wash
or bath, through vapor deposition (e.g., physical or chemical-vapor
deposition), etc. These compounds may also be combined with the cell
composition before and/or during printing, or may be printed or otherwise
applied to the substrate (e.g., coated) as a separate layer beneath,
above, and/or between cell layers. For example, one such support compound
is a gel having a viscosity that is low enough under the printing
conditions to pass through the nozzle of the printer head, and that can
gel to a stable shape during and/or after printing. Such viscosities are
typically within the range of from about 0.5 to about 50 centipoise, in
some embodiments from about 1 to about 20 centipoise, and in some
embodiments, from about 1 to about 10 centipoise. Some examples of
suitable gels that may be used in the present invention include, but are
not limited to, agars, collagen, hydrogels, etc. One example of a
collagen gel for facilitating cell growth is described in Collagen As a
Substrate for Cell Growth and Differentiation, Methods in Enzymology,
Strom and Michalopoulous, Vol. 82. 544-555 (1982) (T. Boland at para 50).

[0032]Besides gels, other support compounds may also be utilized in the
present invention. Extracellular matrix analogs, for example, may be
combined with support gels to optimize or functionalize the gel. One or
more growth factors may also be introduced in the printed cell arrays.
For example, slow release microspheres that contain one or more growth
factors in various concentrations and sequences may be combined with the
cell composition to accelerate and direct the cell fusion process. Other
suitable support compounds might include those that aid in avoiding
apoptosis and necrosis of the developing structures. For example,
survival factors (e.g., basic fibroblast growth factor) may be added. In
addition, transient genetic modifications of cells having antiapoptotic
(e.g., bcl-2 and telomerase) and/or blocking pathways may be included in
cell aggregates to be printed according to the invention. Adhesives may
also be utilized to assist in the survival of the cells after printing.
For instance, soft tissue adhesives, such a cyanoacrylate esters, fibrin
sealant, and/or gelatin-resorcinol-formaldehyde glues, may be utilized to
inhibit nascent constructs from being washed off or moved following
printing of a layer. In addition, adhesives, such as
arginine-glycine-aspartic acid ligands, may enhance the adhesion of cells
to a gelling polymer or other support compound. In addition,
extracellular proteins, extracellular protein analogs, etc., may also be
utilized (T. Boland at para 55).

[0033]Besides two-dimensional arrays, three-dimensional arrays may also be
formed. Three-dimensional cell arrays are commonly used in tissue
engineering and biotechnology for in-vitro and in-vivo cell culturing. In
general, a three-dimensional array is one which includes two or more
layers separately applied to a substrate, with subsequent layers applied
to the top surface of previous layers. The layers can, in one embodiment,
fuse or otherwise combine following application or, alternatively, remain
substantially separate and divided following application to the
substrate. Three-dimensional arrays may be formed in a variety of ways in
accordance with the present invention. For example, in one embodiment,
three-dimensional arrays may be formed by printing multiple layers onto
the substrate. (T. Boland at para 60).

[0034]The thickness of a printed layer (e.g., cell layer, support layer,
etc.) may generally vary depending on the desired application. For
example, in some embodiments, the thickness of a layer containing cells
is from about 2 micrometers to about 3 millimeters, and in some
embodiments, from about 20 micrometers to about 100 micrometers. Further,
as indicated above, support compounds, such as gels, are often used to
facilitate the survival of printed cells. The present inventors have
discovered that the development of a cellular assembly may be increased
when the thickness of the support layer(s) (e.g., between cells) is
approximately the same as the size of the cells deposited adjacent to the
support compound (T. Boland at para 61).

[0035]When printing certain types of two-dimensional or three-dimensional
arrays, it is sometimes desired that any subsequent cell growth is
substantially limited to a predefined region. Thus, to inhibit cell
growth outside of this predefined region, compounds may be printed or
otherwise applied to the substrate that inhibit cell growth and thus form
a boundary for the printed pattern. Some examples of suitable compounds
for this purpose include, but are not limited to, agarose, poly(isopropyl
N-polyacrylamide) gels, and so forth. In one embodiment, for instance,
this "boundary technique" may be employed to form a multi-layered,
three-dimensional tube of cells, such as blood vessels. For example, a
cell suspension may be mixed with a first gel ("Gel A") in one nozzle,
while a second gel ("Gel B") is loaded into another nozzle. Gel A induces
cell attachment and growth, while Gel B inhibits cell growth. To form a
tube, Gel A and the cell suspension are printed in a circular pattern
with a diameter and width corresponding to the diameter and wall
thickness of the tube, e.g., from about 3 to about 10 millimeters in
diameter and from about 0.5 to about 3 millimeters in wall thickness. The
inner and outer patterns are lined by Gel B defining the borders of the
cell growth. For example, a syringe containing Gel A and "CHO" cells and
a syringe containing Gel B may be connected to the nozzle. Gel B is
printed first and allowed to cool for about 1 to 5 minutes. Gel A and CHO
cells are then printed on the agarose substrate. This process may be
repeated for each layer. (T. Boland at para 62).

[0036]The present invention particularly provides for the printing of
tissues by the appropriate combination of cell and support material, or
two or three or more different cell types typically found in a common
tissue, preferably along with appropriate support compound or compounds,
and optionally but preferably with one or more appropriate growth
factors. Cells, support compounds, and growth factors may be printed from
separate nozzles or through the same nozzle in a common composition,
depending upon the particular tissue (or tissue substitute) being formed.
Printing may be simultaneous, sequential, or any combination thereof.
Some of the ingredients may be printed in the form of a first pattern
(e.g., an erodable or degredable support material), and some of the
ingredients may be printed in the form of a second pattern (e.g., cells
in a pattern different from the support, or two different cell types in a
different pattern). Again the particular combination and manner of
printing will depend upon the particular tissue. Materials to be printed
for specific tissues or tissue substitutes are described further below.

[0037]Skin. In representative embodiments, to produce epidermal-like skin
tissue, the following are printed: [0038](a) at least one cell type,
and preferably at least two or in some embodiments three or four
different epidermal cell types (e.g., keratinocytes, melanocytes, Merkel
cells, Langerhan cells, etc., and any combination thereof); and/or
[0039](b) at least one support compound such as described above (e.g.,
collagen, elastin, keratin, etc., and any combination thereof); and/or
[0040](c) at least one growth factor as described above (e.g., basic
fibroblast growth factor (bFGF), Insulin-Like Growth Factor 1, epidermal
growth factor (EGF), etc., and any combination thereof);

[0041]In some embodiments the epidermal cells, support compound and/or
growth factors printed as described above (which form an "epidermal" type
layer) are printed on, or on top of, a previously formed (e.g., printed
or ink-jet printed) "dermal" type layer, the previously printed dermal
layer layers comprising: (a) one, two, three or four different dermal
cells (fibroblasts, adipocytes, mast cells, and/or macrophages), (b) at
least one support compound as described above; and/or (c) at least one
growth factor as described above.

[0042]Skin tissue produced by the process of the present invention is
useful for implantation into or on a subject to, for example, treat
burns, and other wounds such as incisions, lacerations, and crush
injuries (e.g., postsurgical wounds, and posttraumatic wounds, venous leg
ulcers, diabetic foot ulcers, etc.)

[0043]Bone. In particular embodiments, to produce bone tissues, the
following are printed: [0044](a) at least one bone cell type, and
preferably at least two or three different bone cell types (e.g.,
osteoblasts, osteoclasts, osteocytes, and any combination thereof, but in
some embodiments at least osteoblasts and osteoclasts, and in some
embodiments all three); and/or [0045](b) at least one support compound
such as described above (e.g., collagen, hydroxyapatites, calicite,
silica, ceramic, proteoglycans, glycoproteins, etc., and any combination
thereof); and/or [0046](c) at least one growth factor (e.g., bone
morphogenetic protein, transforming growth factor, fibroblast growth
factors, platelet-derived growth factors, insulin-like growth factors,
etc., and any combination thereof).

[0047]Bone tissues produced by the processes described herein are useful
for, among other things, implantation into a subject to treat bone
fractures or defects, and/or promote bone healing.

[0052]Pancreatic islet tissue produced by the processes described herein
is useful for, among other things, implantation into a subject to treat
diabetes (including type I and type II diabetes).

[0053]Nerve. In representative embodiments, to produce nerve tissue, the
following are printed: [0054](a) at least one, two or three cells
types, and preferably (i) a central or peripheral nerve cells (e.g.,
cortical neurons, hippocampal neurons, dopaminergic neurons, cholinergic
neurons, adrenergic neurons, noradrenergic neurons, etc., including any
combination thererof), and/or (ii) at least one glial cell type (e.g.,
neuroglia, astrocytes, oligodendrocytes, Schwann cells, etc., including
any combination thereof) and (iii) any combination thereof (e.g. a
combination of at least one nerve cell and at least one glial cell);
and/or [0055](b) at least one support compound such as described above;
(e.g., laminin, collagen type IV, fibronectin, etc., and any combination
thereof); and/or [0056](c) at least one growth factor (e.g., NGF,
brain-derived neurotrophic factor, insulin-like growth factor-I,
fibroblast growth factor, etc., or any combination thereof); and any
combination of the foregoing.

[0057]Nerve tissue produced by the processes described herein is useful,
among other things, for implantation into a subject to treat nerve injury
or degenerative diseases such as Parkinson's disease and Alzheimer's
disease.

B. Stem Cells.

[0058]In some embodiments stem cells are printed onto substrates by
ink-jet printing. Stem cells may be printed alone (typically in
combination with a support compound or compounds) or in combination with
one or more additional cells (e.g. in a combination selected to produce a
tissue as described above).

[0059]Stem cells (such as pluripotent or multipotent cells) are capable of
differentiating into multiple different cell types or lines, including at
least one of a hepatogenic-specific (or liver-specific) cell line, a
myogenic (or muscle specific) cell line, an osteogenic (or bone specific)
cell line, or an endothelial specific cell line. Useful cells for
carrying out the invention include but are not limited to embryonic stem
cells, parthenogenetic stem cells, amniotic fluid stem cells, and
adipose-derived stem cells.

[0060]Embryonic stem cells useful for carrying out the present invention
are known and described in, for example, U.S. Pat. No. 6,200,806 to
Thomson and U.S. Pat. No. 5,843,780 to Thomson.

[0061]Adipose-derived stem cells are known and described in, for example,
U.S. Pat. No. 6,777,231 to Katz et al.

[0064]In general, AFSCs are cells, or progeny of cells, that are found in
or collected primarily from mammalian amniotic fluid, but may also be
collected from mammalian chorionic villus or mammalian placental tissue.
The cells are preferably collected during the second trimester of
gestation. In mice the cells are most preferably collected during days 11
and 12 of gestation. Preferably the mammalian source is of the same
species as the mammalian subject being treated.

[0065]In general, the tissue or fluid can be withdrawn by amniocentesis,
punch-biopsy, homogenizing the placenta or a portion thereof, or other
tissue sampling techniques, in accordance with known techniques. From the
sample, stem cells or pluripotent cells may be isolated with the use of a
particular marker or selection antibody that specifically binds stem
cells, in accordance with known techniques such as affinity binding
and/or cell sorting. Particularly suitable is the c-Kit antibody, which
specifically binds to the c-kit receptor protein. C-kit antibodies are
known (see, e.g., U.S. Pat. Nos. 6,403,559, 6,001,803, and 5,545,533).
Particularly preferred is the antibody c-Kit(E-1), a mouse monoclonal IgG
that recognizes an epitope corresponding to amino acids 23-322 mapping
near the human c-kit N-terminus, available from Santa Cruz Biotechnology,
Inc., 2145 Delaware Avenue, Santa Cruz, Calif., USA 95060, under catalog
number SC-17806).

[0067]AFSCs also have substantial proliferative potential. For example,
they proliferate through at least 60 or 80 population doublings or more
when grown in vitro. In preferred embodiments AFSCs used to carry out the
invention proliferate through 100, 200 or 300 population doublings or
more when grown in vitro. In vitro growth conditions for such
determinations may be: (a) placing of the amniotic fluid or other crude
cell-containing fraction from the mammalian source onto a 24 well Petri
dish containing a culture medium [α-MEM (Gibco) containing 15%
ES-FBS, 1% glutamine and 1% Pen/Strept from Gibco supplemented with 18%
Chang B and 2% Chang C from Irvine Scientific], upon which the cells are
grown to confluence, (b) dissociating the cells by 0.05% trypsin/EDTA
(Gibco), (c) isolating an AFSC subpopulation based on expression of a
cell marker c-Kit using mini-MACS (Mitenyl Biotec Inc.), (d) plating of
cells onto a Petri dish at a density of 3-8×103/cm2, and
(e) maintaining the cells in culture medium for more than the desired
time or number of population doublings.

[0068]Preferably, the AFSCs are also characterized by the ability to be
grown in vitro without the need for feeder cells (as described in PCT
Application WO 03/042405 to Atala and DeCoppi. In preferred embodiments
undifferentiated AFSCs stop proliferating when grown to confluence in
vivo.

[0069]AFSCs used to carry out the present invention are preferably
positive for alkaline phosphatase, preferably positive for Thy-1, and
preferably positive for Oct4, all of which are known markers for
embryonic stem cells, and all of which can be detected in accordance with
known techniques. See, e.g., Rossant, J., Stem cells from the Mammalian
blastocyst. Stem Cells, 2001. 19(6): p. 477-82; Prusa, A. R., et al.,
Oct-4-expressing cells in human amniotic fluid; a new source for stem
cell research? Hum Reprod, 2003.18(7): p. 1489-93.

[0070]In a particularly preferred embodiment, the AFSCs do not form a
teratoma when undiferentiated AFSCs are grown in vivo. For example,
undifferentiated AFSCs do not form a teratoma within one or two months
after intraarterial injection into a 6-8 week old mouse at a dose of
5×106 cells per mouse.

[0072]In preferred embodiments the amniotic fluid stem cells used to carry
out the present invention do not express CD34 and CD105, markers of
certain lineage restricted progenitors, nor the hematopoietic marker
CD45.

[0073]In preferred embodiments the amniotic fluid stem cells used to carry
out the present invention express low levels of major histocompatibility
(MHC) Class I antigens and are negative for MHC Class II.

[0074]Differentiation of cells. "Differentiation" and "differentiating" as
used herein include (a) treatment of the cells to induce differentiation
and completion of differentiation of the cells in response to such
treatment, both prior to printing on a substrate, (b) treatment of the
cells to induce differentiation, then printing of the cells on a
substrate, and then differentiation of the cells in response to such
treatment after they have been printed, (c) printing of the cells,
simultaneously or sequentially, with a differentiation factor(s) that
induces differentiation after the cells have been printed, (d) contacting
the cells after printing to differentiation factors or media, etc., and
combinations of all of the foregoing. In some embodiments differentiation
may be modulated or delayed by contacting an appropriate factor or
factors to the cell in like manner as described above. In some
embodiments appropriate differentiation factors are one or more of the
growth factors described above. Differentiation and modulation of
differentiation can be carried out in accordance with known techniques,
as described in greater detail below, or as described in U.S. Pat. No.
6,589,728, or US Patent Application Publication Nos.: 2006006018
(endogenous repair factor production promoters); 20060013804 (modulation
of stem cell differentiation by modulation of caspase-3 activity);
20050266553 (methods of regulating differentiation in stem cells);
20050227353 (methods of inducing differentiation of stem cells);
20050202428 (pluripotent stem cells); 20050153941 (cell differentiation
inhibiting agent, cell culture method using the same, culture medium, and
cultured cell line); 20050131212 (neural regeneration peptides and
methods for their use in treatment of brain damage); 20040241856 (methods
and compositions for modulating stem cells); 20040214319 (methods of
regulating differentiation in stem cells); 20040161412 (cell-based VEGF
delivery); 20040115810 (stem cell differentiation-inducing promoter);
20040053869 (stem cell differentiation); or variations of the above or
below that will be apparent to those skilled in the art.

[0075]Pancreas. Differentiation of cells to pancreatic-like cells can be
carried out in accordance with any of a variety of known techniques. For
example, the cells can be contacted to, printed with, or cultured in a
conditioning media such as described in US Patent Application
2002/0182728 (e.g., a medium that comprises Dulbecco's Minimal Essential
Medium (DMEM) with high glucose and sodium pyruvate, bovine serum
albumin, 2-mercaptoethanol, fetal calf serum (FCS), penicillin and
streptomycin (Pen-Strep), and insulin, transferrin and selenium). In
another example, the cells may be treated with a cAMP upregulating agent
to induce differentiation as described in U.S. Pat. No. 6,610,535 to Lu.
In still another example, the cells may be grown in a reprogramming
media, such as described in US Patent Application 2003/0046722A1 to
Collas to induce differentiation to a pancreatic cell type. In another
embodiment, differentiation may be carried out using the 5 steps protocol
describe by Lumelsky at al. Lumelsky, N., et al., Differentiation of
embryonic stem cells to insulin-secreting structures similar to
pancreatic islets. Science, 2001. 292(5520): p. 1389-94. In another
embodiment, differentiation may be carried out using DMSO to induce
pancreatic differentiation in vitro. She-Hoon Oh et al, Adult bone
marrow-derived cells trans-differentiating into insulin-producing cells
for the treatment of type I diabetes. Lab Inv, 2004, 84: 607-617. In
another embodiment, differentiation may be carried out using Nicotinamide
to induce pancreatic differentiation in vitro. See, e.g., Otonkoski, T.,
et al, Nicotinamide is a potent inducer of endocrine differentiation in
cultured human fetal pancreatic cells. J Clin Invest, 1993, 92(3):
1459-1466. In another embodiment, differentation may be carried out using
inhibitors of phosphoinositide 3-kinase (PI3K), such as LY294002, to
induce pancreatic differentiation in vitro. See, e.g., Hori, Y., et al.,
Growth inhibitor promote differentiation of insulin-producing tissue from
embryonic stem cells. PNAS, 2002, 99:16105-16110. In another embodiment
Exendin-4, a naturally occurring 39-amino acid peptide originally
isolated from the salivary secretions of the Lizard Heloderma suspectum,
can be used to induce pancreatic differentiation in vitro. Nielsen, LL.,
et al., Pharmacology of exenatide (synthetic exendin-4): a potential
therapeutic for improved glycemic control of type 2 diabetes. Regul Pept.
2004 Feb. 15; 117(2):77-88. Review. In still another embodiment,
anti-sonic hedgehog (Anti-Shh) and co-culturing with pancreatic rudiments
can be used to induce pancreatic differentiation in vitro. Leon-Quinto,
T., et al., In vitro direct differentiation of mouse embryonic stem cells
into insulin-producing cells. Diabetologia, 2004, 47:1442-1451. In one
preferred embodiment the differenting step is carried out by transducing
(sometimes also referred to as "engineering" or "transforming") the cells
with a vector, or introducing into the cells a vector, that contains a
nucleic acid encoding a differentiation factor (such as Pdx1, Ngn3,
Nkx6.1, Nkx2.1, Pax6, or Pax4) and expresses the differentiation factor
in the cells, or by activating the expression of an endogeneous nucleic
acid encoding a differentiation factor in the cells (e.g., engineering
the cells to activate transcription of an endogeneous differentiation
factor such as Pdx1, Ngn3, Nkx6.1, Nkx2.1, Pax6, or Pax4, such as by
inserting a heterologous promoter in operative associated with an
endogeneous differentiation factor, in accordance with known techniques.
See, e.g., U.S. Pat. No. 5,618,698). Such exogeneous nucleic acids may be
of any suitable source, typically mammalian, including but not limited to
rodent (mouse, hamster, rat), dog, cat, primate (human, monkey), etc.

[0078]Myogenic induction: Cells may be induced to promote myogenic
induction by any suitable technique, such as culturing in myogenic medium
(DMEM low glucose supplemented with 10% horse serum, and 0.5% chick
embryo extract from Gibco) followed by treatment of 5-azacytidine (10
μM, Sigma) added in myogenic medium for 24 h.

[0079]Endothelial induction: Cells may be induced to promote endothelial
induction by any suitable technique, such as culturing in endothelial
basal medium-2 (EBM-2, Clonetics BioWittaker) supplemented with 10% FBS
and 1% glutamine (Gibco).

[0080]The present invention is explained in greater detail in the
following non-limiting examples.

Example 1

Printing of Multiple Cell Types

[0081]Materials and Methods. Three distinct cell types were used in this
study: human amniotic fluid-derived stem cells (hAFSC) transfected with
lacZ, bladder smooth muscle cells (BSMC), and GFP labeled MS1 (mouse
pancreatic islet endothelial cell line). Each cell type was grown
separately, trypsinized, collected and resuspended in Type I collagen
solution. Different mixtures of collagen and cells were loaded into
different ink cartridges. Each cell-collagen mixture was printed
layer-by-layer into the pre-designed target locations using a modified HP
550 printer. A solution containing NaOH was subsequently printed in order
to neutralize the pH. The printed constructs were placed in the incubator
for 3-5 hours. Once the collagen gel was set, 3-D viable multi-cellular
constructs with a specific shape were formed. After 2 days of culture,
the printed multi-cellular constructs were fixed and characterized using
cell specific markers (α-actin, X-gal).

[0082]To examine the function of each cell type within the printed
constructs, hAFSC cells were induced to differentiate into osteogenic
lineage followed by evaluation of calcium production using Alizarin red
staining. Smooth muscle cell function was assessed by measuring the
resting membrane potentials and K.sup.+ currents using a patch clamp
system (Axopatch 200B).

[0083]Results and Discussion. Fabrication of multi-cellular structures.
All three printed cell types were confirmed by their corresponding cell
identification methods, as shown in FIG. 1. The GFP labeled MS1 cells
exhibited green fluorescence and smooth muscle cells emitted red under
UV. The X-gal staining confirmed the lacZ transfected hAFSC cells in blue
under bright field microscopy. All three cell types were present in an
organized fashion within the printed construct. A 3-D collagen "pie" with
different color dyes was shown in FIG. 1E, demonstrating the capability
of the inkjet printers to print different biomaterials as well as
multiple cell types.

[0084]Functional evaluation. Alizarin red staining showed the production
of calcium in the osteogenic differentiation culture of hAFSC (FIG. 2a),
which suggests that hAFSC in the collagen constructs retain their
capability to differentiate into specific cell lineages under appropriate
conditions. The whole cell patch clamp recording showed the average
resting membrane potential of the printed BSMC (-58.5±5.8 mV), which
is similar to normal non-printed smooth muscle cells (-54.7±7.5 mV).
There was no significant difference on the K.sup.+ I-V relationship
between the printed cells and the normal controls (FIG. 2b). These
findings demonstrate that smooth muscle cells in the printed collagen
constructs maintained their normal basic electrophysiological properties.

[0085]Conclusions. This example shows that viable three-dimensional
heterogeneous constructs with multiple cell types can be generated by
printing multiple cells and collagen gels layer-by-layer. These distinct
cells are able to survive and proliferate within the 3-D constructs, and
maintain normal basic cellular properties and function in their spatially
registered regions. These findings demonstrate the possibility of
building complex tissues that require multiple cell types and ECM
materials by using the bio-printing technology.

Example 2

In vivo Generation of Tissues with Ink-Jet Printing

[0086]In this example we investigated whether the printed multi-cell
derived tissue constructs could maintain their structural and spatial
orientation in vivo. We examined whether these tissues are able to
survive and mature into functional tissues when implanted in vivo.

[0087]Materials and Methods: Three-dimensional multi-cell constructs with
a "pie" configuration were fabricated by simultaneously printing 3
different cell types [canine bladder smooth muscle cells (SMC), bovine
aortal endothelial cells (EC), and human amniotic fluid-derived stem
cells (AFSC)] into collagen/alginate gel. The cells were labeled with 3
different membrane bound tracers, which include [PKH67 (red), PKH26
(green), and CMHC (blue)], respectively, prior to printing. Individual
cells were also printed separately for additional testing. The printed 3D
constructs were subcutaneously implanted into athymic mice. AFSC-printed
constructs were cultured in osteogenic medium for 1 week before
implantation in order to induce differentiation into bone tissue. The
implanted constructs were monitored by MRI and micro-CT scanner over time
(up to 18 weeks). The retrieved engineered tissues were analyzed with
confocal microscopy and immunohistochemical studies. To evaluate the
function of the engineered muscle, electrophysiological properties were
performed with voltage clamp experiments.

[0090]In vivo functional evaluation. The voltage clamp recording showed
that the printed SMCs exhibited similar patterns in the mean current
voltage (I-V) relationships before and after implantation (FIG. 4a),
which suggests that the SMCs are able to maintain normal basic
electrophysiological characteristics in vivo. Vascularization of the
EC-printed implants was evaluated by MRI scanning 8 weeks
post-implantation. After the gadolinium (Gd) contrast agent was injected
intravenously into the animal, contrast enhancement was visualized within
the implants, which indicates the presence of vascular network within the
implanted tissues. FIG. 4b shows intensified MRI signals, and the degree
of contrast enhancements is denoted in different colors in the implants.
The formation of vascularization was reconfirmed by the presence of blood
vessels, which were positively expressed with the endothelial
cell-specific marker: vWF (FIG. 4d). These data suggest that EC-printed
implants are able to form functional vasculature. Micro CT scanning
showed that bone-like hard tissues were formed within the AFSC-printed
constructs 18 weeks post-implantation (FIG. 4c). Immuonohistochemical
analysis showed that the differentiated AFSCS within the implant
expressed a typical bone cell marker, osteocalcin (FIG. 3d). These data
suggest that the printed stem cells within the constructs retain their
ability to differentiate into specific cell lineages and enhance
formation of relevant tissues under specific conditions.

[0091]Conclusions. This example shows that multi-cellular constructs,
generated by the inkjet method, are able to maintain their structural and
spatial orientation in vivo. The printed cells are able to retain their
cellular characteristics and tissue function. The inkjet printing
technology may become a standard method of engineering functional tissues
for clinical applications.

[0092]The foregoing is illustrative of the present invention, and is not
to be construed as limiting thereof. The invention is defined by the
following claims, with equivalents of the claims to be included therein.